I have been committed to a career in basic science since my first exposure to research as an undergraduate student. After completing an MD/PhD program, I went on to pursue surgical residency and then a fellowship in abdominal transplant surgery. To achieve my goal of becoming a successful independent researcher, I recently entered the laboratory of Dr. John Engelhardt, a national authority on the molecular pathophysiology of cystic fibrosis (CF) and strategies to utilize recombinant adenovirus-associated viruses (rAAV) as gene therapy vectors. Dr. Engelhardt has proven track records in both mentoring physician- scientists and securing research funding, and his guidance will be a critical determinant of my success in developing my independent research laboratory. Under Dr. Engelhardt's mentorship, I am focused on combining my basic science training with my medical background to tackle an important clinical question that directly impacts my patients, namely studying the pathophysiology of cystic fibrosis-related diabetes (CFRD). This is a clinically important question, as CF is the most common lethal autosomal recessive disease in Caucasians, resulting from defects in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. This K-award will allow critical protected time for me to fully develop my independent research career. During the period of this award, I intend to: 1) expand my skills in molecular genetics, 2) master the techniques relevant to CF research and learn to generate pathophysiologically important hypotheses about CFRD, 3) develop skill in the implementation of rAAV gene therapies, 4) strengthen my writing and leadership/mentoring skills, and 5) solidify preliminary data for my first R01 application. In CF patients, pancreatic disease leads to diabetes mellitus in up to 50% of adult patients, resulting in reduced life expectancy. Progress in elucidating the pathophysiology of CFRD has been hindered by the lack of an animal model. Dr. Engelhardt's laboratory has recently developed a CF-knockout ferret - the first animal model that develops CFRD. However, CF ferrets have high perinatal mortality from abnormal intestinal and pancreatic function, altered glucose and lipid metabolism, and lung infections. Elevating GI pH appears to attenuate nutritional abnormalities, as the use of proton-pump inhibitors (PPIs) normalizes weight gain during the neonatal period. The CF ferret model appears to be a robust system for the development of CF pancreatic therapies and is the only animal model available for the study of CFRD. We hypothesize that effective treatment of CFRD will lead to reduced nutritional complications in CF ferrets, increased long-term survival, and reduced severity of lung disease. We will test this hypothesis by carrying out the following specific aims: 1) Identify the defects underlying the pancreatic and intestinal pathophysiologies associated with CFRD in the ferret CF model and 2) Establish a CFTR gene replacement therapy that will improve pancreatic function in CF ferrets. To address the goals of Aim 1, we will use standard pancreatic endocrine functional tests to confirm that a subset of CF ferrets develop CFRD and characterize the disease pathophysiology. We will determine if CFTR-mediated bicarbonate (HCO3-) secretion by the pancreatic duct and/or duodenal epithelial cells is reduced, and if so, how this impacts CFRD phenotypic severity. We will also evaluate the activity of compensatory, non-CFTR-based HCO3- secretory pathways using CFTR inhibitors to determine if these channels might influence the severity and/or disease onset of CFRD. To address the goals of Aim 2, we will adapt methods developed for the mouse (application of rAAV vectors encoding the WT ferret CFTR cDNA) for in vivo gene delivery to the pancreas in CFTR-/- ferrets. We will assess the effectiveness of pancreatic CFTR gene replacement in the CFTR-/- ferrets by evaluating serum glucose, insulin, glucagon, endocrine pancreatic function, and pancreatic duct cell physiology. We will also assess the impact of pancreatic CFTR gene therapy by evaluating ferret growth and nutritional parameters in the presence and absence of PPIs and pancreatic enzymes. Finally, we will evaluate the impact of pancreatic CFTR gene therapy on the progression of early pulmonary disease progression in CF ferrets following removal of antibiotics. These studies will assess lung inflammatory mediators (IL-1, IL-8, and TNFa), lung bacterial colonization, and lung histopathology. The major expected outcomes of this study are: 1) a more comprehensive characterization of endocrine pancreas abnormalities in the new CF ferret model, 2) an understanding of the basis of phenotypic variation in CFTR-associated pancreatic disease observed in both the CF ferrets and humans, and 3) the development of a pancreatic gene replacement approach that will aid in dissecting pathophysiology and treatment of CFRD. These results are expected to have a significant positive impact on the field of CF research. PUBLIC HEALTH RELEVANCE: Cystic fibrosis (CF) is the most common life-threatening autosomal recessive condition among Caucasians, with over $450 million dollars spent annually on clinical care of CF patients in the U.S. alone. In CF patients, pancreatic disease leads to diabetes mellitus in up to 50% of adult patients over the age of 30 years, resulting in reduced life expectancy. Progress in elucidating the pathophysiology of CF-related diabetes (CFRD) has been hindered by the lack of appropriate animal models for use in studying the disease process and testing therapies. Using a new CF animal model of CFRD, this proposal will characterize pathophysiologic mechanisms of disease and develop gene therapies to treat pancreatic disease in the model.